The division of chromosomes into euchromatic and heterochromatic regions is perhaps the most striking and enigmatic aspect of genome organization in multicellular eukaryotes. Heterochromatin is a major component of higher eukaryotic genomes as diverse as humans and fruit flies, and it plays important roles in chromosome inheritance and metabolism. However, large gaps exist in our understanding of the structure of heterochromatin, because this region is rich in repetitive sequences. Without a detailed understanding of heterochromatin structure, genome function and evolution will remain obscure. We have used the molecular genetic tools available in Drosophila to elucidate basic information on the structure and sequence composition of heterochromatin. We have cloned and partially sequenced a 420 kb minichromosome functional centromere, and have generated single copy entry points (P transposable elements) that will facilitate structural and functional studies of the rest of Drosophila heterochromatin. In collaboration with the Berkeley Drosophila Genome Center (BDGP), we have demonstrated that a new Whole Genome Shotgun sequence reliably assembles a significant fraction of the Drosophila heterochromatin. We annotated 12 Mb of this heterochromatic sequence, producing approximately 450 heterochromatic gene models, and revealing important data on the organization and repeat content of Drosophila heterochromatin. Here we propose to create the Drosophila Heterochromatin Genome Project (DHGP), whose goal is to obtain a more detailed understanding of the sequence composition and organization of Drosophila centric heterochromatin. Our goals are to generate and annotate approximately 18 Mb of finished heterochromatic sequence. Our strategy integrates existing approaches, tools and reagents available for Drosophila genome studies. This comprehensive approach is essential to a successful analysis of these 'difficult' genomic regions. The results of this study will elucidate the sequence organization and composition of Drosophila heterochromatin. The project will also identify and analyze heterochromatic genes, paving the way to understanding their organization, regulation and function. These studies will provide information and tools that will further our understanding of higher eukaryotic genome structure, and will lay the groundwork for more complete analyses of heterochromatin structure and function in Drosophila and other eukaryotes, including humans.